Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa - PubMed (original) (raw)

doi: 10.1101/gr.086082.108. Epub 2008 Dec 1.

Morgan G I Langille, Joanne L Fothergill, Irena Kukavica-Ibrulj, Catherine Paradis-Bleau, François Sanschagrin, Nicholas R Thomson, Geoff L Winsor, Michael A Quail, Nicola Lennard, Alexandra Bignell, Louise Clarke, Kathy Seeger, David Saunders, David Harris, Julian Parkhill, Robert E W Hancock, Fiona S L Brinkman, Roger C Levesque

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Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in the Liverpool Epidemic Strain of Pseudomonas aeruginosa

Craig Winstanley et al. Genome Res. 2009 Jan.

Abstract

Pseudomonas aeruginosa isolates have a highly conserved core genome representing up to 90% of the total genomic sequence with additional variable accessory genes, many of which are found in genomic islands or islets. The identification of the Liverpool Epidemic Strain (LES) in a children's cystic fibrosis (CF) unit in 1996 and its subsequent observation in several centers in the United Kingdom challenged the previous widespread assumption that CF patients acquire only unique strains of P. aeruginosa from the environment. To learn about the forces that shaped the development of this important epidemic strain, the genome of the earliest archived LES isolate, LESB58, was sequenced. The sequence revealed the presence of many large genomic islands, including five prophage clusters, one defective (pyocin) prophage cluster, and five non-phage islands. To determine the role of these clusters, an unbiased signature tagged mutagenesis study was performed, followed by selection in the chronic rat lung infection model. Forty-seven mutants were identified by sequencing, including mutants in several genes known to be involved in Pseudomonas infection. Furthermore, genes from four prophage clusters and one genomic island were identified and in direct competition studies with the parent isolate; four were demonstrated to strongly impact on competitiveness in the chronic rat lung infection model. This strongly indicates that enhanced in vivo competitiveness is a major driver for maintenance and diversifying selection of these genomic prophage genes.

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Figures

Figure 1.

Figure 1.

Circular map of the P. aeruginosa LES genome. Starting from outermost circle going inwards: major (500 kb) and minor tick (100 kb) measurements of the genome with estimated location of the origin; prophage (orange) and genomic islands (green) are highlighted across all tracks; protein coding genes (blue) on plus (outer) and minus strand (inner); tRNAs (green), rRNAs (orange), and all other noncoding RNA genes (purple); signature tagged mutants (black); GC content (outer black line plot) with GC content average (gray line); and GC skew (inner black line plot) were calculated using a 10-kb non-overlapping window. Note that one region of low GC, upstream of the first noted prophage, plus additional smaller regions of low GC, contain ribosomal genes that are commonly known to have a lower GC in genomes. The location of two highly similar genomic regions of length 7.5 kb and 13.5 kb within the prophages are marked with looping purple lines, between their locations on the innermost circle. The identified prophage and GIs are distributed around the genome, but there is one notable cluster of LESGI-1, LESGI-2, and LESGI-3, reflecting the nonrandom nature of GI insertion in P. aeruginosa (Wiehlmann et al. 2007). Significant sequence composition bias in seven of the nine regions was computationally identified (Table 2), while GC content deviating from the average can be observed for these regions in the figure.

Figure 2.

Figure 2.

Phage clusters identified in LESB58 with significant similarities and positioning of STM mutants after in vivo screening.

Figure 3.

Figure 3.

Genomic islands identified in LESB58 with significant similarities and positioning of STM mutants after in vivo screening.

Figure 4.

Figure 4.

In vivo competitive index (CI) of the P. aeruginosa STM PALES_45041 (within LESGI-5), PALES_25621 (within LES prophage 5), PALES_13261 (within LES prophage 3), and PALES_08021 (within LES prophage 2) grown for 7 d in the rat lung in competition with the wild-type LESB58 strain. Each circle represents the CI for a single animal in each group. A CI of less than 1 indicates an attenuation of virulence. The geometric mean of the CIs for all rats is shown as a solid line and statistically significant _P_-value is indicated with an asterisk (*P < 0.001 using the Mann-Whitney sum test).

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